Kit for detecting fcv and fhv and use

Abstract

The application relates to a kit for detecting FCV and FHV and application, the kit comprising: a PCR reaction solution, the PCR reaction solution comprising an FCV primer probe group, an FHV primer probe group, an enzyme, a stabilizer and water; a nucleic acid releasing solution, the nucleic acid releasing solution comprising a surfactant, an alkali agent, a chelating agent and a lysis enhancer; wherein the FCV comprises forward primers FCVF and reverse primers FCVR shown as SEQ ID NO:1 and SEQ ID NO:2; and the FHV primer probe group comprises forward primers FHVF and reverse primers FHVR shown as SEQ ID NO:4 and SEQ ID NO:5. The kit can quickly, conveniently and accurately detect cat cup and cat herpesvirus type I, thereby being applied to the detection of FCV and FHV.

Description

Technical Field

[0001] This application relates to a kit and its application for detecting FCV and FHV, belonging to the field of nucleic acid molecular detection. Background Technology

[0002] Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the main pathogens causing respiratory diseases in cats and are viruses that cause upper respiratory tract infections (URIs) in cats.

[0003] Traditional detection methods, particularly those involving nucleic acid extraction, mostly employ magnetic bead extraction and column extraction. These processes are cumbersome and time-consuming. The extensive mechanical handling can generate significant amounts of nucleic acid fragments, leading to aerosol contamination. Furthermore, these methods place high demands on testing reagents, equipment, and personnel. Pet hospitals, requiring immediate testing and rapid results, also fall short of the needs of such end-user scenarios. Therefore, current detection methods are not well-suited to the requirements of pet hospitals and similar facilities.

[0004] Furthermore, regarding sample addition, pipettes are routinely used. However, veterinary hospital staff, lacking professional training, are prone to using pipettes without adjusting the volume, leading to inaccurate sample volumes and experimental failures. Additionally, pipette tips are mostly provided in boxes, and veterinary hospitals cannot maintain cleanliness by constantly using and closing them, making contamination highly likely.

[0005] Based on the above situation, there is an urgent need for a new method to detect feline calicivirus and feline herpesvirus type I, in order to provide technical support for clinical diagnosis and treatment. Summary of the Invention

[0006] The purpose of this application is to provide a kit and application for detecting FCV and FHV, which can quickly, conveniently and accurately detect feline calicivirus and feline herpesvirus type I.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] In a first aspect, this application provides a kit for detecting FCV and FHV, comprising:

[0009] PCR reaction solution, wherein the PCR reaction solution comprises FCV primer and probe set, FHV primer and probe set, enzyme, stabilizer and water;

[0010] Nucleic acid release solution, wherein the nucleic acid release solution comprises surfactant, alkali, chelating agent and lysis enhancer;

[0011] The FCV primer-probe set includes the forward primer FCVF and the reverse primer FCVR as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe FCVP; the FHV primer-probe set includes the forward primer FHVF and the reverse primer FHVR as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe FHVP.

[0012] In one or more feasible embodiments, the sequence of the FCVP is as shown in SEQ ID NO:3.

[0013] In one or more feasible embodiments, the sequence of the FHVP is as shown in SEQ ID NO:6.

[0014] In one or more feasible embodiments, the PCR reaction solution further includes an internal standard primer-probe set, which includes the cat housekeeper gene forward primer IF as shown in SEQ ID NO:7, the cat housekeeper gene reverse primer IR as shown in SEQ ID NO:8, and the cat housekeeper gene probe primer IP as shown in SEQ ID NO:9.

[0015] In one or more feasible embodiments, the 5' and 3' ends of the FCVP, FHVP, and IP are labeled with a fluorescent group and a quenching group, respectively, and the fluorescent groups labeled on the FCVP, FHVP, and IP are all different.

[0016] In one or more feasible embodiments, the fluorescent group may be any one of HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, Alexa Flour, or VIC.

[0017] In one or more feasible embodiments, the quenching group may be any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3 or QSY4.

[0018] In one or more feasible embodiments, the enzyme includes DNA polymerase, thermosensitive reverse transcriptase, and anti-contamination enzyme.

[0019] In one or more feasible embodiments, the stabilizer is selected from Tween 20, glycerol, BSA, ammonium sulfate, and DTT.

[0020] In one or more feasible embodiments, the PCR reaction solution comprises 10-200 mM Tris-HCl, 10-100 mM KCl, 1-6 mM MgCl2, 0.2-0.6 mM dNTPs, 0.1-0.8 µm of FCVF / FCVR / FCVP / FHVF / FHVR / FHVP, 0.05-0.4 µm of IF / IR / IP, 0.1%-1% Tween 20, 1-5% glycerol, 0.05-1% BSA, 1-20 mM ammonium sulfate, 1-5 mM DTT, 0.01-0.1% Proclin 300, 0.5 U-4 U DNA polymerase, 20-100 U thermosensitive reverse transcriptase, UDG enzyme, and DEPC water.

[0021] In one or more feasible embodiments, the surfactant is selected from sodium dodecylbenzenesulfonate, CHAPS, or a combination thereof.

[0022] In one or more feasible embodiments, the alkaline agent is sodium hydroxide.

[0023] In one or more feasible embodiments, the chelating agent is disodium ethylenediaminetetraacetate.

[0024] In one or more feasible embodiments, the pyrolysis enhancer comprises sodium tripolyphosphate and aminotriacetic acid.

[0025] In one or more feasible embodiments, the nucleic acid release solution comprises 1-100 mM potassium chloride, 0.1-1% aminotriacetic acid, 0.01%-2% 3-[(3-Cholanidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.05-1M sodium hydroxide, 0.05%-2% sodium tripolyphosphate, 0.01-1% sodium dodecylbenzenesulfonate, and 1-50 mM disodium ethylenediaminetetraacetate.

[0026] In one or more feasible implementations, the kit is used for rapid fluorescent PCR reactions.

[0027] In one or more feasible embodiments, the kit further includes a disposable sample loop having a sampling end and a handheld end, the radial dimension of which gradually decreases from the handheld end to the sampling end.

[0028] Secondly, this application provides the use of the kit described in the first aspect in the detection of FCV and FHV.

[0029] In one or more feasible embodiments, the application includes the preparation of rapid PCR nucleic acid amplification reaction tubes.

[0030] In one or more feasible implementations, the application includes using a disposable sampling loop having a sampling end and a handheld end for sample application.

[0031] In one or more feasible embodiments, the radial dimension of the disposable sample loop gradually decreases from the handheld section to the sampling end.

[0032] Through the implementation of this application, the beneficial effects of this application are as follows:

[0033] The reagent kit described in this application eliminates the need for complex procedures such as magnetic bead extraction, high-temperature lysis, and column centrifugation in its nucleic acid release solution. The designed primer-probe set exhibits low dimer counts, and the combination of thermosensitive reverse transcriptase and dual-antibody DNA polymerase plays a crucial role in the stability of the reaction solution. Furthermore, the PCR reaction solution contains stabilizers, enhancing the room temperature and 2–8°C stability of the fully premixed reaction solution, saving preparation time, and reducing losses due to incorrect storage or handling.

[0034] In other words, the kit provided in this application has high specificity and broad coverage for detecting feline calicivirus and herpes simplex. Compared with the group standard, its sensitivity is higher: 20 copies / test. Furthermore, using the kit for rapid PCR reactions is not only simple to operate but also features an anti-contamination system and a loop-based error-proofing mechanism. More importantly, the application is time-efficient, taking only 45 minutes; it is low-cost, high-throughput, and applicable to multiple scenarios, meeting not only hospital needs but also those for catteries and pet import / export testing.

[0035] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, the preferred embodiments of this application are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of PCR detection of feline calicivirus according to one embodiment of this application;

[0037] Figure 2 This is a schematic diagram of PCR detection of feline herpesvirus type I according to one embodiment of this application;

[0038] Figure 3 This is a schematic diagram of the structure of a disposable sample loop shown in one embodiment of this application. Detailed Implementation

[0039] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, unless otherwise specified, the following embodiments and features in the embodiments can be combined with each other.

[0040] It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of this application. Therefore, the illustrations only show the components related to this application and are not drawn according to the number, shape and size of the components in actual implementation. In actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0041] Some exemplary embodiments of this application have been described for illustrative purposes. It should be understood that this application may be implemented in other ways not specifically shown in the accompanying drawings.

[0042] This application provides a kit for detecting FCV and FHV, primarily for rapid PCR reactions, comprising:

[0043] PCR reaction solution, wherein the PCR reaction solution comprises FCV primer and probe set, FHV primer and probe set, enzyme, stabilizer and water;

[0044] Nucleic acid release solution, wherein the nucleic acid release solution comprises surfactant, alkali, chelating agent and lysis enhancer;

[0045] The FCV primer-probe set includes the forward primer FCVF and the reverse primer FCVR as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe FCVP; the FHV primer-probe set includes the forward primer FHVF and the reverse primer FHVR as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe FHVP.

[0046] In at least one embodiment of this application, the sequence of the FCVP is shown in SEQ ID NO:3, and the sequence of the FHVP is shown in SEQ ID NO:6.

[0047] By way of example and not limitation, the PCR reaction solution also includes an internal standard primer and probe set, which includes the cat housekeeper gene forward primer IF as shown in SEQ ID NO:7, the cat housekeeper gene reverse primer IR as shown in SEQ ID NO:8, and the cat housekeeper gene probe primer IP as shown in SEQ ID NO:9.

[0048] The applicant found through comparison that using the above three sets of primer and probe sets reduced the dimer at the 3' end, effectively preventing non-specific generation and playing an important role in the stability of the system.

[0049] Those skilled in the art will understand that FCVP, FHVP, and IP are labeled with a fluorescent group and a quenching group at their 5' and 3' ends, respectively, and the fluorescent groups labeled on FCVP, FHVP, and IP are all different. For example, the fluorescent group can be any one of HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, Alexa Flour, or VIC. The quenching group can be any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3, or QSY4.

[0050] In one embodiment of this application, the 5' end of the FCVP probe is labeled with the FAM fluorescent group and the 3' end with the MGB quencher group; the 5' end of the FHVP probe is labeled with the VIC fluorescent group and the 3' end with the BHQ1 quencher group; the 5' end of the IP primer is labeled with the roX fluorescent group and the 3' end with the BHQ2 quencher gene.

[0051] As an example, and not a limitation, the enzymes in the PCR reaction solution include DNA polymerase, thermosensitive reverse transcriptase, and anti-contamination enzyme. The added stabilizers are selected from one or more of Tween 20, glycerol, BSA, ammonium sulfate, and DTT.

[0052] In at least one embodiment of this application, the PCR reaction solution comprises 10-200 mM Tris-HCl, 10-100 mM KCl, 1-6 mM MgCl2, 0.2-0.6 mM dNTPs, 0.1-0.8 µm of FCVF / FCVR / FCVP / FHVF / FHVR / FHVP, 0.05-0.4 µm of IF / IR / IP, 0.1%-1% Tween 20, 1-5% glycerol, 0.05-1% BSA, 1-20 mM ammonium sulfate, 1-5 mM DTT, 0.01-0.1% Proclin 300, 0.5 U-4 U DNA polymerase, 20-100 U thermosensitive reverse transcriptase, UDG enzyme, and DEPC water.

[0053] By way of example and not limitation, the surfactant is selected from sodium dodecylbenzenesulfonate, CHAPS, or combinations thereof. For example, the alkali is sodium hydroxide, the chelating agent is disodium ethylenediaminetetraacetate, and the pyrolysis enhancer includes sodium tripolyphosphate and aminotriacetic acid.

[0054] In at least one embodiment of this application, the nucleic acid release solution comprises 1-100 mM potassium chloride, 0.1-1% aminotriacetic acid, 0.01%-2% CHAPS, 0.05-1M sodium hydroxide, 0.05%-2% sodium tripolyphosphate, 0.01-1% sodium dodecylbenzenesulfonate, and 1-50 mM disodium ethylenediaminetetraacetate.

[0055] Most commercially available products store enzyme solutions and reaction solutions separately, or provide reaction solutions in a lyophilized form. This solves the problem of transportation and preservation, and reduces the risk of reagent failure due to dimerization. However, an additional mixing or reconstitution step is required for end-user use, increasing the number of steps. For veterinary hospital staff who have not received professional PCR training, this increases the possibility of contamination and operational errors.

[0056] The PCR reaction solution mentioned above is a fully premixed reagent that can be stable at room temperature for 3 days and at 2~8℃ for 7 months, saving solution preparation time and reducing losses caused by incorrect storage or operation, which meets the needs of end customers.

[0057] The applicant discovered that the aforementioned kit can be widely used in the detection of FCV and FHV. Specifically, the sample to be tested is added to the nucleic acid release solution of the kit, and then a sampling tool is used to transfer the sample from the nucleic acid release solution to the PCR reaction solution, followed by a rapid PCR reaction. Finally, the Ct value is used to determine the positive or negative result: the internal standard channel (ROX channel) must have a clear amplification curve; if the FAM channel has a clear amplification curve, it is considered feline cupid's disease positive; if the VIC channel has a clear amplification curve, it is considered feline herpesvirus positive.

[0058] Regarding sample addition, pipettes are routinely used. However, veterinary hospital staff, lacking professional training, are prone to using pipettes directly without adjusting the volume, leading to inaccurate sample volumes and experimental failures. Furthermore, pipette tips are mostly provided in boxes, and veterinary hospitals rarely maintain the cleanliness of these tips by immediately closing them after use, making them susceptible to contamination.

[0059] In at least one embodiment of this application, the sampling tool is a disposable sampling loop.

[0060] The present application will be further described in detail below with reference to specific embodiments.

[0061] PCR reaction solution

[0062] The plasmids constructed from the conserved sequences of the feline cupella and feline herpes genes were quantified using Qubit and serially diluted to 2000 copies / μL. 2 μL of the FCV / FHV plasmid was added to the reaction solution under the conditions shown in Table 1. Additional 0.1 mM dNTPs, 2.5 U DNA polymerase, 20 U thermosensitive reverse transcriptase, 1 U thermosensitive UNG enzyme, and the concentrations of FCVF / FCVR / FCVP (250 nM), FHVF / FHVR / FHVP (250 nM), and IF / IR / IP (150 nM) were added. The final volume was brought to 18 μL with DEPC water.

[0063] Table 1

[0064] Serial Number Tris-HCl pH 8.5 KCl <![CDATA[MgCl2]]> 1 100 mM 50 mM 3 mM 2 150 mM 50 mM 3 mM 3 200 mM 50 mM 3 mM 4 150 mM 100 mM 3 mM 5 150 mM 200 mM 3 mM 6 150 mM 50 mM 4 mM 7 150 mM 50 mM 5 mM

[0065] The fluorescence amplification curves of the PCR reaction system for FCV and FHV are shown in the figure. Figure 1 and Figure 2 As shown in the figure, the PCR reaction solution system of group 7 performed best in both FCV and FHV detection.

[0066] Clinical sample testing

[0067] Because feline calicivirus is an RNA virus with a very high mutation rate, clinical samples were used to verify whether the selected primers and probes could effectively identify real samples. The primers for the control group were based on the group standard T / CVMA 38-2020 published by the Chinese Veterinary Medical Association.

[0068] Samples: Twenty eye, nose and mouth swabs with respiratory symptoms were collected from a pet hospital. Two swabs were collected from each sample.

[0069] The experimental group used a one-step release method. Twenty eye, mouth, and nose swabs were directly inserted into 1 mL of the release solution, the swabs were broken, and the cotton tip remained in the solution. The cap was screwed on, the mixture was shaken well, and after standing at room temperature for 3 minutes, 2 μL was added to the reaction solution for instrumental analysis. The release solution consisted of: 100 mM potassium chloride, 0.5% aminotriacetic acid, 0.05% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 0.1 M sodium hydroxide, 0.1% sodium tripolyphosphate, 0.05% sodium dodecylbenzenesulfonate, and 5 mM disodium ethylenediaminetetraacetate solution.

[0070] The control group used the magnetic bead method. Twenty swab samples were first soaked in 1 ml of physiological saline for 5 min, then 200 μL was extracted, eluted with 200 μL of DEPC water, and then 2 μL was added to the reaction solution for instrument detection. The results are shown in Table 2.

[0071] Table 2

[0072] Sample number Experimental group - cup-shaped Control group - cup 1 Undetect Undetect 2 33.8 36.1 3 25.8 27.6 4 34.3 36.5 5 35.2 36.83 6 Undetect Undetect 7 27.9 26.3 8 35.5 Undetect 9 31.2 30.4 10 30.1 36.5 11 Undetect Undetect 12 34.0 35.2 13 Undetect Undetect 14 Undetect Undetect 15 27.1 34.2 16 22.9 27.9 17 Undetect Undetect 18 34.9 38.8 19 Undetect Undetect 20 Undetect Undetect

[0073] As shown in Table 2, the positive rates of the primers in the experimental group and the control group were basically the same, and the Ct values ​​were also higher. This indicates that the primers selected in the experimental group can effectively identify cat cup primers, and also shows that the one-step release method has high sensitivity.

[0074] Rapid PCR reaction program

[0075] Clinically positive feline calicivirus samples with a Ct value of approximately 30 were selected as the calicivirus screening sample, and 40 copies / μL (2μL) of FCV / FHV plasmid were selected as the feline herpes screening sample. Amplification and validation were performed using the procedures shown in Table 3, with 40 cycles and each procedure repeated 10 times.

[0076] Table 3

[0077]

[0078] The cup-shaped results are shown in Table 4. As can be seen from Table 4, the results obtained by using the conditions of program 1 are the best, programs 2 and 4 are basically the same, and programs 3 and 5 are slightly worse, indicating that the reverse transcription process of more than 3 minutes has a better effect.

[0079] The results for the herpes zoster test are shown in Table 5. As can be seen from Table 5, except for a slight delay in Ct in procedure 3, the other groups were basically consistent, indicating that an extension time of more than 15 seconds can meet our requirements. Combining the experimental results of the caliciform and herpes zoster groups, we chose procedure 2, which has a relatively shorter time, to meet market demand without affecting the judgment of experimental results.

[0080] Table 4

[0081] Serial Number Program 1 Program 2 Program 3 Program 4 Program 5 1 30.26 31.36 31.34 31.47 32.13 2 30.27 32.13 32 31.50 34.62 3 31.11 32.03 31.08 30.66 32.90 4 31.13 31.8 34.28 31.30 32.00 5 30.02 31.2 32.61 32.17 33.33 6 31.22 31.04 32.17 31.90 33.14 7 31.44 31.9 33.38 32.61 34.82 8 31.21 31.09 32.21 32.01 32.87 9 30.25 32.17 34.30 32.48 33.27 10 31.25 31.15 32.17 31.11 32.10 mean 30.81 31.58 32.55 31.72 33.11 CV 1.76% 1.46% 3.41% 1.96% 2.95%

[0082] Table 5

[0083] Serial Number Program 1 Program 2 Program 3 Program 4 Program 5 1 34.14 33.82 35.72 34.59 35.41 2 33.53 33.49 34.7 35.24 35.97 3 33.54 34.92 35.7 34.45 34.46 4 34.01 34.46 34.68 35.15 34.85 5 33.52 34.01 35.62 34.14 34.19 6 33.47 34.87 36.03 34.56 34.35 7 34.08 33.49 34.8 34.32 33.91 8 33.84 34.53 34.79 35.29 35.35 9 34.43 34.08 35.12 34.06 34.50 10 33.84 34.8 36.46 35.2 34.12 mean 33.84 34.247 35.362 34.7 34.711 CV 0.96% 1.59% 1.78% 1.38% 1.92%

[0084] Sensitivity verification of disposable sample loops

[0085] The FCV / FHV plasmid was diluted to 10 copies / μL as a sensitivity reference. The experimental group used disposable sample loops for sample loading. The structure of the disposable sample loop is shown below. Figure 3 It has a sampling end and a handheld end. The radial dimension of the disposable sampling ring gradually decreases from the handheld end to the sampling end. Furthermore, the sampling end has a ring-shaped structure with a through hole in the middle to facilitate the passage of liquid.

[0086] The control group used a pipette to aspirate 2 μL of sample. This was repeated 20 times. The results are shown in Table 6. All results were positive, indicating that the sensitivity of the pipette loop was comparable to that of the pipette.

[0087] Table 6

[0088]

[0089] stabilizer

[0090] The PCR reaction solution additives were prepared as shown in Table 7. The fully premixed reaction solutions were stored at 2–8°C and 25°C, respectively. The samples were moderately positive clinical samples, lysed using a one-step release buffer, and three tests were repeated. The mean value was taken. The control group had no additives. The enzyme solution and reaction solution were stored separately at -20°C. During testing, 17.4 μL of reaction solution + 0.6 μL of enzyme solution were mixed. The results are shown in Table 8. Formulas 3 and 4 were relatively stable, but formula 4 showed a delayed Ct value compared to the control group initially.

[0091] Table 7

[0092] Serial Number glycerin Tween20 BSA ammonium sulfate DTT Proclin300 1 0% 0% 0% 0mM 0mM 0% 2 2% 0.05% 0.05% 10mM 1mM 0.05% 3 4% 0.1% 0.1% 20mM 5mM 0.05% 4 8% 0.5% 0.5% 50mM 10mM 0.05%

[0093] Table 8

[0094]

[0095] Based on the above embodiments, in terms of the reagent kit, the nucleic acid release solution provided in this application does not require magnetic bead extraction, high-temperature lysis, column centrifugation and other complex operations, and can effectively release viral nucleic acid in just 5 minutes or even less. It is easy to operate, requires no professional training, and reduces the contamination of nucleic acid fragments.

[0096] Furthermore, according to the embodiments of this application, three sets of primer probes are preferred, and primer probes for monitoring the cat housekeeper gene are added, which can effectively monitor the sampling and amplification process; and by different combinations, combinations with fewer dimers are selected, and thermosensitive reverse transcriptase and double-antibiotic DNA polymerase are used in conjunction, which play an important role in the stability of the reaction solution.

[0097] Meanwhile, this application optimizes the reaction solution additives, improving the room temperature stability and 2-8℃ stability of the fully premixed reaction solution, saving solution preparation time, and reducing losses due to incorrect storage or handling, thus meeting the needs of end customers. The use of the UNG enzyme system effectively eliminates contamination from environmental products, making it more suitable for the use cases of small pet hospitals.

[0098] In terms of operation, this application uses disposable sample loops, simplifying the sample addition process and reducing the possibility of errors and contamination. The combination of nucleic acid release solution, rapid PCR program, and disposable sample loops makes the operation simple and takes approximately 45 minutes, meeting the needs of end-user hospitals.

[0099] In summary, the kit provided in this application exhibits high specificity and broad coverage for detecting feline calicivirus and herpes simplex. Compared to the group standard, its sensitivity is higher at 20 copies / test. Furthermore, the rapid PCR reaction using this kit is not only simple to operate but also features an anti-contamination system and a loop-based error-proofing mechanism. Crucially, the application is time-efficient, taking only 45 minutes; it is low-cost, high-throughput, and versatile, suitable not only for hospital use but also for catteries and pet import / export testing.

[0100] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0101] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. The application of a kit for detecting FCV and FHV in the preparation of products for detecting FCV and FHV, characterized in that, The application includes the preparation of rapid PCR nucleic acid amplification reaction tubes, the product includes PCR nucleic acid amplification reaction tubes and disposable sample loops for sample addition, the disposable sample loops having a sampling end and a handheld end, and the kit includes: The PCR reaction solution is a fully premixed liquid reagent, comprising FCV primer and probe sets, FHV primer and probe sets, enzymes, stabilizers, and water; wherein the enzymes include DNA polymerase, thermosensitive reverse transcriptase, and anti-contamination enzyme; the stabilizers include 4% glycerol, 0.1% Tween 20, 0.1% BSA, 20mM ammonium sulfate, 5mM DTT, and 0.05% Proclin 300; A nucleic acid release solution, comprising a surfactant, an alkali, a chelating agent, and a lysis enhancer; wherein the nucleic acid release solution comprises 100 mM potassium chloride, 0.5% aminotriacetic acid, 0.05% CHAPS, 0.1M sodium hydroxide, 0.1% sodium tripolyphosphate, 0.05% sodium dodecylbenzenesulfonate, and 5 mM disodium ethylenediaminetetraacetate; The FCV primer-probe set includes the forward primer FCVF and the reverse primer FCVR as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe FCVP; the FHV primer-probe set includes the forward primer FHVF and the reverse primer FHVR as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe FHVP; the sequence of FCVP is shown in SEQ ID NO:3, and the sequence of FHVP is shown in SEQ ID NO:

6.

2. The application as described in claim 1, characterized in that, The PCR reaction solution also includes an internal standard primer and probe set, which includes the cat housekeeper gene forward primer IF as shown in SEQ ID NO:7, the cat housekeeper gene reverse primer IR as shown in SEQ ID NO:8, and the cat housekeeper gene probe primer IP as shown in SEQ ID NO:

9.

3. The application as described in claim 2, characterized in that, The 5' and 3' ends of the FCVP, FHVP, and IP are labeled with fluorescent groups and quenching groups, respectively, and the fluorescent groups labeled on the FCVP, FHVP, and IP are all different.

4. The application as described in claim 1, characterized in that, The kit is used for rapid fluorescent PCR reaction; the disposable sample loop has a sampling end and a handheld end, and the radial dimension of the disposable sample loop gradually decreases from the handheld end to the sampling end.

5. The application as described in claim 1, characterized in that, The radial dimension of the disposable sample application loop gradually decreases from the handheld section to the sampling end.

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